A dynamic, complex relationship exists between stem cells and their microenvironment, which plays a pivotal role in cell fate determination. Key to identifying the molecular mechanisms underlying stem cell plasticity, is understanding the unique epigenetic role of the microenvironment on the emergence of cell phenotype. Previous studies from our laboratory have revealed the unexpected finding that metastatic human melanoma cells express multiple cellular phenotypes and their respective precursor cells, suggesting a dedifferentiated cancer cell with a phenotype characteristic of stem cells. Additional evidence supporting the concept of tumor cell plasticity includes: the demonstration of human metastatic melanoma cells forming vasculogenic- like networks with the simultaneous expression of endothelial-specific genes; developing chimeric blood vessels in an ischemic mouse limb model; and initiating the formation of tissues in an embryonic zebrafish model. Furthermore, recent preliminary findings indicate the powerful influence of a metastatic melanoma microenvironment with respect to inducing transdifferentiation of normal melanocytes into an aggressive tumor cell phenotype ~ when exposed to this metastatic milieu. Most interestingly, preliminary findings with human embryonic stem cells (hESCs) exposed to a metastatic microenvironment reveal their differentiation to a highly migratory phenotype; while the microenvironment associated with hESCs reverts amelanotic metastatic melanoma cells to a melanocyte-like phenotype with pigmentation. Therefore, based on these intriguing observations, we propose to test the central hypothesis that the microenvironment associated with stem cells -- representative of hESCs. cancer stem cells, and normal epidermal stem cells -- contains informational cues with the potential to epigeneticallv reprogram the genotype and phenotype of cells exposed to it. Using unique. 3-D organotypic models and zebrafish embryos, together with functional analysis, imaging, laser microdissection. and global gene analysis, we propose to: Aim 1: Determine the comparative epigenetic effects of the 3-D microenvironments associated with hESCs, cancer stem cells (primarily melanoma), and normal epidermal stem cells for their potential to reprogram the genotype and phenotype of specific stem cell populations. Aim 2: Identify the molecular basis for the epigenetic reprogramming of the genotype and phenotype of the affected stem cell populations exposed to various 3-D microenvironments. Aim 3: Investigate the developmental plasticity of the embryonic, cancer and normal epidermal stem cells in an embryonic zebrafish model - to determine the biological relevance of key regulatory pathways involved in the control of cellular phenotype and cell fate determinations. At the completion of these studies, we expect to gain novel insights into the differences in inductive properties of the respective microenvironment(s) of these stem cells that could be translated for novel therapeutic targets.